Rapid Detection For Aeromonas Salmonicida And Aptamer Screening Of Two Pathogenic Bacteria In Fish

Aeromonas salmonicida and A.hydrophila are the main pathogens of fish in marine and lake ecosystems,which can cause furunculosis,hemorrhagic septicemia and even death in fish,as well as human disease through water and food.Therefore,it is significant for aquaculture,public health and safety to establish techniques and methods for fast detection of these pathogens in water.Aptamer is a single-stranded DNA?ssDNA?or RNA sequence which was obtained by systematic evolution of ligands by exponential enrichment?SELEX?technique.Because of its advantages in high specificity,high affinity and strong stability,aptamer has been widely used in disease diagnosis,biosensor,targeted therapy and other fields.As a new type of polymer nano-materials,graphite-like carbon nitride?g-C₃N₄?has showed a great application potential in the fields of biosensor and biological imaging due to its advantages in continuous luminescence,non-toxicity,excellent biological compatibility and adsorption of ssDNA.In this thesis,a specific aptamer against A.salmonicida was successfully obtained by Cell-SELEX.Combined with the unique properties of g-C₃N₄,a ratio fluorescence nano-biosensor was established and applied to the rapid detection of A.salmonicida in water.At the same time,the aptamer with specific binding to A.hydrophila flagellin FlgE was also screened by SELEX.The main works are summarized as follows:?1?Screening the aptamer for A.salmonicida.A group of aptamers specifically binding to A.salmonicida were obtained by Cell-SELEX.After high-throughput sequencing and homology analysis,three aptamers sequences were selected to identify their binding properties.Among them,the aptamer A.s-2 had the highest binding effect?Kd=41.78±3.6 nM?and specificity combining A.salmonicida.?2?Construction of a ratio fluorescence sensor based on graphite-like carbon nitride for detection of A.salmonicida.A ratio fluorescence nano-biosensor was constructed of the nucleic acid fragments containing the capture function of aptamer A.s-2 linked to the DNAzyme sequences as probes,followed by non-covalent binding to g-C₃N₄ nanosheets.The results indicated that the sensing strategy had high sensitivity toward A.salmonicida in a range of 10³?10⁷ CFU/mL?R²=0.9655?with a detection limit of 10³ CFU/mL.Besides,the proposed method has been successfully applied to the detection of A.salmonicida in diseased zebrafish.Thus,the system may provide an efficient and rapid method for disease diagnosis in fish.?3?Selecting the aptamer for A.hydrophila flagellin FlgE.A group of aptamers specifically binding to A.hydrophila flagellin FlgE were obtained by the screening kit of SELEX.After recombinant plasmid pET-28a-flgE was transformed into competent cells,the induced recombinant protein FlgE was purified and concentrated.In addition,a group of aptamers with high specifically binding to the protein FlgE were selected by the screening kit of SELEX.Among them,the selected aptamer A.h-1 had the highest affinity?Kd=40.3±2.8 nM?and specificity toward A.hydrophila through high-throughput sequencing,affinity and specificity analysis.Therefore,the results laid the foundation for the subsequent development of aptamer-based sensor for the detection of A.hydrophila.